Anti-paratopic antibody as an immunogen

ABSTRACT

The present invention provides a method of manufacture of an anti-paratopic antibody comprising the steps of: (1) selecting from a pool of antibodies occurring in one species a prototypic set the members of which are effective in binding a specific antigen (or antigen epitope), and (2) utilizing one or more members of said prototypic set, or paratopic fragments thereof, as an immunogen in a host of a different species, or in an in vitro incubation system comprising cells derived from the same or a different species, to produce antibodies having a characteristic which is anti-paratopic with respect to said immunogen to produce a synthetic replicate of the specific antigen or epitope. Antigen (or antigen epitope), and monoclonal antibodies. vaccines and processes of immunization employing the product of the method of manufacture are also described.

FIELD OF THE INVENTION

[0001] This invention relates to immunology and more particularly to amethod of manufacture of immunogenic, compositions, to immunogensmanufactured by the method, and to antibodies manufactured therefrom.

BACKGROUND ART

[0002] An immunogen is a molecule capable of eliciting an immuneresponse in a vertebrate. The response elicited is believed to bedetermined by topographical shape characteristics of the immunogen.Immunogens are also called antigens i.e. ANTIbody GENerators because oneaspect of the induced response involves the production of antibodymolecules whose function is to lock onto the immunogen. Those areas ofthe immunogen to which the antibody molecule binds are variouslyreferred to as the antigenic determinants, epitopes or haptens. The lastterm, namely hapten, is generally associated with the term carrier andthis term refers to that part of the immunogen/antigen which interactswith cellular components of the vertebrate immune system.

[0003] These regions on the immunogen and the names used to define themshould not be regarded as absolute. Thus the genus of vertebrates hasimmune systems which will recognize immunogens; but not all speciesnecessarily recognize the same molecular areas as being haptenic areasor carrier areas. Within a species, the recognition of haptenicmolecular areas can only be determined experimentally. Thus, mice willnot necessarily process immunogens in the same way as would, forexample, the immune system of Man. Furthermore, within a species,individual specimens will not respond to the same degree. This isbecause the immune response to an immunogen has a genetic (hereditary)component. Thus, some individuals will respond better to an immunogenwhile others may not respond at all.

[0004] The immune response to an immunogen is an integrated phenomenonin that a class of white blood cells called T lymphocytes, for example,reacts with the carrier determinants which in turn allows a class ofwhite blood cells called B lymphocytes to transform and start producingantibodies to the antigenic determinants.

[0005] Each cell recognizes only one determinant and each antibodyproducing B cell (plasma cell) generates only antibody molecules of onegiven specificity. Hence, the immune system is said to be highlyspecific. Upon stimulation, these plasma cells multiply and thereby giverise to a clone of identical antibody secreting cells. If it werepossible to isolate these identical antibody secreting cells, they wouldbe referred to as monoclonal and the antibodies referred to asmonoclonal antibodies.

[0006]FIG. 1 is a diagrammatic illustration of the response of a mouseto an immunogen/antigen. Under normal conditions each monoclonalantibody generated by the mouse in vivo mixes with other monoclonalantibodies so that a polyclonal antibody response eventuates.

[0007] Each antibody comprises a glycoprotein molecule. The portion ofan antibody molecule embodying the characteristic of shape or moleculartopography, or code sequence which enables it to bind and so for exampleneutralise the antigenic determinant or epitope of an antigen is knownas a “paratope”. The paratope is conceptually a molecular region of ashape complimentary to the epitope or to a part of the epitope of theantigen and is thought to reside in the so called hypervariable regionof the antibody glycoprotein molecule.

[0008] Antibody producing lymphocytes are present in high concentrationin the spleen but antigen reactive spleen lymphocytes cannot readily becultured in isolation. However, monoclonal antibodies may bemanufactured and isolated therefrom by use, for example, of techniquesof hybridoma technology. In one such technique mice are first exposed toan antigen whereby the mouse develops antibodies. With reference to FIG.2, spleen cells of the immunised mouse are fused with mouse myelomacells. The growth of hybrid cells is promoted and the hybrids arescreened for specific antibody secretion. Those useful are cultured orundergo further genetic stabilisation procedures. By this means specificmonoclonal antibodies may be produced and isolated.

[0009] Selected antibodies, or mixtures thereof such as are produced inthe method of FIG. 2, may be used to neutralise an antigen in anorganism, a paratope of each antibody in effect forming a complex withan epitope of the antigen.

[0010] In anti-idiotypic immunology a second stage process shown in FIG.3 is involved. Mouse 1 is first immunised with an antigen. Therebygiving rise to several clones of antibody producing cells. One cell lineis chosen on the basis of the characteristics of the generated antibodyand the antibody is referred to as Ab1. Ab1 is then used to immunise asecond mouse—mouse 2. The latter must have a genetic constitution verysimilar to, or identical with that of mouse 1. Mouse 2 generatesmonoclonal antibodies to Ab1, a subset of which may be directed againstthe paratope of Ab1. All the antibody subsets generated by mouse 2against Ab1 may be referred to as Ab2 though the Ab2 subset specific forthe paratope of subset 1 is sometimes referred to as Ab2 beta. Thesecond mouse monoclonal antibody, Ab2, has an anti-paratope, that is tosay having a molecular portion with a shape characteristic complementaryto the paratope of the first antibody. If the epitope of the originalantigen is considered to be “mould positive”, the paratope of themonoclonal antibody Ab1 can be considered to be a counterpart or “mouldnegative” and the paratope of the anti-Ab1 antibody that is the paratopeof the Ab2 monoclonal antibody can be considered to replicate the “mouldpositive”. It will be understood however that in each case thereplication is not exact. When used in a vaccine, the second monoclonalantibody, Ab2, functions as a harmless immunogen which stimulatesproduction of AB3 antibodies in the vaccinated animal effectivelyproducing immunity to the first antigen.

[0011] Anti-idiotypic vaccines are designed to be an interspeciesapproach so as to identify epitopes which induce neutralising antibodiesin genetically diverse population. The approach requires that theanti-idiotypic vaccine candidates (Ab2) be inoculated into populationsas diverse as sheep, chimpanzees and rabbits followed by antigenchallenge to determine if the Ab3 carries the neutralisingcharacteristics of the Ab1. If the challenge is successful Ab1 isfurther studied as a possible vaccine. If challenge is unsuccessful, theantibody is disregarded for its role as a vaccine. In this approach, theanimal studies are meant to simulate the genetic diversity found in Man.The epitopes are therefore exclusively public in that they are capableof generating neutralizing responses in a number of different species.

DISCLOSURE OF THE INVENTION

[0012] According to one aspect of the present invention consists of amethod of treating an animal comprising the steps of:

[0013] (1) selecting from a pool of antibodies occurring in a firstspecies of vertebrate a prototypic set the members of which areantibodies with specificity for a specific antigen or antigen epitopeexpressed in one or more members of the first species, and not expressedin members of a second species;

[0014] (2) utilizing one or more members of said prototypic set, orparatopic fragments thereof, as an immunogen in a host of the secondspecies, wherein said second species is selected such that the immunesystem of said second species does not recognise the specific antigen orantigen epitope uniquely expressed in one or more members of said firstspecies, or said paratopic set or paratopic fragments thereof areutilized as an immunogen in an in vitro incubation system comprisingcells derived from the same or a different species, to produceantibodies having a characteristic which is anti-paratopic with respectto said immunogen to produce a synthetic replicate of the specificantigen or epitope.

[0015] (3) introducing anti-paratopic antibodies produced in step 2 intothe same or a different members of the first species selected in step 1.

[0016] According to a second aspect the present invention consists in amethod of manufacture of an anti-paratopic antibody efficacious againstmammalian infections comprising the steps of:

[0017] (1) selecting from a pool of antibodies occurring in a firstspecies of vertebrate a prototypic set the members of which areantibodies with specificity for a specific antigen expressed in one ormore members of said first species and not expressed in members of asecond species;

[0018] (2) utilizing one or more members of said prototypic set, orparatopic fragments thereof, as an immunogen in a host of the secondspecies wherein said second species is selected such that the immunesystem of said second species does not recognise the specific antigen orantigen epitope uniquely expressed in one or more members of said firstspecies, or said paratopic set or paratopic fragments thereof areutilized as an immunogen in an in vitro incubation system comprisingcells derived from the same or a different species to produce one ormore antibodies having a characteristic which is anti-paratopic withrespect to the antigen or antigen epitope expressed in one or moremembers of said first species but not expressed in members of the secondspecies; and

[0019] (3) selecting and purifying said anti-paratopic antibodiesproduced according to step 2.

[0020] In a preferred embodiment of the invention the anti-paratopicmonoclonal antibodies are used to immunize a different member of thefirst species as that from which the prototypic set was selected.

[0021] For preference the pool of antibodies consists of naturallyoccurring human antibodies.

[0022] The prototypic set is a set of antibodies selected on the basisof effectiveness against a particular antigen, or epitope thereof, forexample is a set of human antibodies selected from humans carryingantibodies resulting from exposure to HIV.

[0023] The antibodies, or paratypic paratope bearing segments of them,are utilized as a immunogen in a mouse host to produce mouse antibodieshaving anti-paratope characteristics.

[0024] The mouse antibodies are then screened first for their ability tospecifically bind antigens or antigen epitopes in relation to the firstspecies and then in respect of their effectiveness for inducingantibodies which bind the HIV in other members of the first species.

[0025] It will be understood that the response generated against theantigen or antigen epitope under consideration would be uncommon innearly all species except the first species from which the prototypicset of antibodies was selected.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 is a diagrammatic illustration of the response of a mouseto an immunogen/antigen.

[0027]FIG. 2 is a diagrammatic representation of monoclonal antibodyproduction.

[0028]FIG. 3 illustrates anti-idiotypic antibody Ab2 production.

[0029]FIG. 4 is a schematic representation of the method of manufactureof anti-paratopic antibodies according to the invention.

[0030]FIG. 5 (I) illustrated a general procedure for the purification ofHIV positive human antibodies.

[0031]FIG. 5 (II) illustrates a general procedure for the purificationof HIV antigen specific human antibodies.

[0032]FIG. 6(a) illustrates purification of human IgG prior todelineation into HIV/HIV antigen specific antibodies.

[0033]FIG. 6(b) illustrates purification of human IgA prior todelineation into HIV/HIV antigen specific antibodies.

[0034]FIG. 6(c) illustrates purification of human IgM prior todelineation into HIV/HIV antigen specific antigens.

[0035] Preferred embodiments of the invention have a number ofadvantages over the prior art.

[0036] Firstly, the invention produces a mouse anti-paratope which is acounterpart of a naturally occurring human antibody paratope for aspecified antigen. Upon inoculation the mouse anti-paratope monoclonalantibody produces in a human an antibody bearing a replica of thenaturally occurring human paratope.

[0037] In the prior art there was produced a mouse anti-paratope whichwas a counterpart of an artificially generated mouse antibody. Such amouse anti-paratope monoclonal antibody would produce in a human anantibody bearing a replica of an artificially created mouse paratope (incontrast to a human paratope) and which may not be as effective inbinding the specific antigen in a human. In relation to the prior artthe invention does not rely upon the assumption inherent within theprior art that the mouse processes antigen in exactly the same way ashumans.

[0038] Secondly, the present invention has as its basic premise theobservation that epitope presentation within a particular species isunique to that species. The prior art technology looks toimmuno-dominant epitopes common to all species and aims to producemirror images to them. Epitopes selected in the prior art technology areidentified using empirical approaches and numerous algorithums have beenused to predict antigenic sequences. In the present invention, antigenicsequences necessary to produce neutralizing epitopes are believed to beboth linear and assembled. Antigen presentation is a multifactorialoperation involving several host immune components. Hence, the basicpremise of the present invention is that epitope mapping algorithmswhile applicable do not identify all epitopes of immunologicalsignificance. It is in this area which the present invention is focused.The host immune system has a role in the amplification and display ofthe total repertorie of epitopes of the invading immunogen. The presentinvention capitalises on this factor whilst prior art technologies havetended to approach the problem from a more conventional anti-idiotypicapproach.

[0039] Thirdly, in comparison with the prior art scheme illustrated inFIG. 3, the invention provides a more direct general route shownschematically in FIG. 4 to the production of an anti-idiotypic antibody.

[0040] Fourthly, since preferred embodiments of the invention use widelyavailable and naturally occurring i.e. endogenous, antibodies as thestarting material rather then antigens, the process is expected to beless costly to conduct.

[0041] Fifthly, the process is safer to conduct than processes requiringhandling for example of potentially harmful virus antigens.

[0042] Finally, both the present invention and anti-idiotypic technologyuse hybridoma technology, protein chemistry and immunology. When testingthe putative vaccine, however, anti-idiotype vaccines have to be testedfor complimentary and efficacy in several animal species e.g. rabbits,sheep, baboons or chimpanzees etc. This is necessary to compensate forthe interspecies approach used to generate and test the vaccinecandidate. This is a relatively long and time consuming step. Thevaccine candidate produced in the present invention, however, has to betested primarily for complementary within the species to be immunized.It is designed to be primarily an intra-species approach. Accordingly,from a small pool of infected individuals either immune to a particularinfectious agent or carrying neutralizing antibodies to it and usingstandard techniques (or minor technological variants) to produceanti-paratopic antibodies, it is possible using the present invention togenerate specific vaccines against said infectious agent. These vaccinescan then be used to treat a small number of infected individuals or toimmunize an entire population of individuals prone to infection by saidinfectious agent.

[0043] The particular advantage of the present invention is that in thecase of some viruses, for example AIDS, there are so many epitopes someof which are protective, some of which are suppressive, some of whichare dominant and some which have no affect on the immune system. In thepresent invention, selection of the antibody is not dependent on theepitope. Instead, selection is based on whether the antibody produced isneutralizing or not. In contrast, in prior art approaches, antibodyselection depends on an epitope being common to a variety of differentspecies. For example, if an antibody works in rabbits but not in guineapigs, it is discarded. The result being to reject it but in doing so,the very epitopes which could protect human populations may be lost.This is overcome by the present invention because reliance is not placedon epitope recognition between species.

BEST MODES FOR PERFORMING THE INVENTION

[0044] An embodiment of the invention will now be described by way ofexample only. The embodiment concerns the manufacture of a vaccine toconfer immunity against Acquired Immune Deficiency Syndrome (AIDS). Theinvention is not, however, limited to use for production of anyparticular vaccine, and has uses other than for the production ofvaccines.

[0045] The manufacture may be considered as involving the steps of:

[0046] (1) selecting a prototypic set of antibodies;

[0047] (2) preparing one or more immunogens therefrom;

[0048] (3) inoculating hosts with one or more immunogens;

[0049] (4) generating a monoclonal antibody pool from each host;

[0050] (5) screening the monoclonal antibody pools;

[0051] (6) testing the screened antibodies for effectiveness as avaccine.

[0052] In the example under consideration the first stage is to selectfrom the pool of human antibodies a prototypic set, in this case a setof immunoglobulins which effectively bind the aetiologic agent forAcquired Immune Deffiency Syndrome (AIDS). The generally acceptedaetiological agent for AIDS is currently known as Human ImmunodeficiencyVirus hereinafter referred to as HIV.

[0053] That is accomplished by obtaining human immunoglobulins fromindividuals exposed to HIV. About 75% of such individuals haveantibodies to HIV.

[0054] The antibodies from these individuals are screened foreffectiveness in binding HIV antigens and/or antigenic fragments. Thoseantibodies effective at this function are retained as members of theprototypic set i.e. they are a subset of the pool of humanimmunoglobulins.

[0055] If desired the retained immunoglobulin members so selected may bepurified an used directly in step (3).

[0056] Preferably, however, in a second step the human immunoglobulins(“Ig”) are subdivided into classes G, A, M, D, E (to use the WHOdesignation) and more particularly identified in Table I. TABLE 1PHYSICAL PROPERTIES OF MAJOR HUMAN IMMUNOGLOBULIN CLASS IN SERUM WHODesignation IgG IgA IgM IgD IgE Sedimentation 75 75,9S,11S* 19S# 75 85Coefficient Molecular 150,000 160,000 + dimer 900,000 185,000 200,000Weight Number of Ig 1 1-2 5 1 1 Units Number of Antigen 2 2-4 10 2 2Binding Sites Identity of (gamma) (alpha) (mu) (delta) (epsilom) HeavyChain Carbohydrates 3  8 12 13 12 Content % Total Immunoglobulin 80 13 60-1 .002 in normal human serum Concentration range 8-16 mg/ml 1.4-4mg/ml 0.5-2 mg/ml 0-0.4 mg/ml 17-450 mg/ml in normal human serum

[0057] More desirably still the immunoglobulins are further divided intosub-classes, for example, IgG being divided into four sub-classes, IgAbeing divided into two sub-classes and IgM into two sub-classes. In thepreferred embodiment each of sub-classes IgG 1-4, IgA 1-2 and IgM 1-2are purified and isolated from each other. IgD and IgE sub-classes arepresent in immunoglobulin in small concentration and their inclusion isoptional.

[0058] The human IgG/A/M is drawn from three main groups affected byAIDS viral infection, viz

[0059] male homosexuals

[0060] bisexual/female/heterosexual AIDS carriers

[0061] haemophilics

[0062] Blood plasma is heated to 56° C. to kill the virus. Cellularcomponents and serum debris are removed either by aspiration of theserum component or by centrifugation (in the case of plasma).

[0063] Human IgG can be purified free of all non-IgG contaminants byaffinity chromatography. Other procedures such as ion-exchangechromatography may be used but affinity chromatography is preferred forspeed and selectivity. More particularly, purification is generallyeffected by means of chromatography using PROTEIN-A SEPHAROSE beads(obtainable from e.g. Pharmacia Biotechnology Pty Ltd.).

[0064] Sub-classes of IgG may also be isolated by chromatography.

[0065] In a similar manner human IgA purification may be carried out byanti-IgA affinity chromatography.

[0066] Human IgM may be purified by a combination of

[0067] (a) Protamine sulphate chromatography, and

[0068] (b) Column chromatography, or

[0069] (c) IgM affinity chromatography.

[0070] The purified prototypic immunoglobulins set may be used directlyas an immunogen for inoculation of mice in stage 3.

[0071] Alternatively, the Ig sub-classes may be screened to selectantigen specific antibodies for use as the immunogen. In this case, theIg sub-classes are next screened for effectiveness against HIV antigento select the most effective sub-classes in binding the antigen. Morepreferably, the antigen is first divided into sub-classes known as p18,p24, gp41, p55, gp120 and gp160. These antigen sub-classes differ fromeach other in molecular structure and can be separated bySDS-polyacrylamide gel electrophoresus. Each Ig sub-class is thenscreened against each antigen sub-class to select the most effectiveIg's. HTLV III - HUMAN SERUM Ig PARATOPE GRID Human Serum Ig's* AntigenIgG (80%) IgA (13%) Specific HTLV III No. of 1 2 3 4 1 2 IgM (6%) HumanAntigens Epitopes (65%) (23%) (8%) (4%) (80%) (20%) 1 2 Paratope p24 216 gp41 4 32 p53 5 40 gp120 12 96 qp160 16 128 Carrier 39 39 39 39 39 3939 39 Specific Idiotypes Total Number 166 78 78 per Ig Class % Spread of50% 25% 25% Idiotype

[0072] With reference to Table 2, there is shown a “paratope grid”. Ifit is assumed that there is one antigenic group anchored to a tenthousand dalton carrier group then the total number of antigenic groups(epitopes) available among the five antigenic sub-classes would bethirty nine. With eight potential antibody classes in the grid that canrespond to the thirty nine antigens the total number of possibleantibodies carrying paratopes specific for HIV is 312. Put differently,there are on average thirty nine HIV paratopic bearing humanimmunoglobulins per immunoglobulin sub-class. Thus, for example, itmight eventuate that human IgG1 has specificities for all thirty nineepitopes (“haptens”), i.e., there would be thirty nine IgG1 moleculesall absolutely identical except for one feature namely their Fabparatope would be different.

[0073] In the third step of the embodiment, one or more members of theprototypic set are used as an immunogen in a non-human host for exampleby being injected into a mouse. The one or more members are preferablythe most effective of the immunoglobulin sub-classes. The criteria ofeffectiveness may be effectiveness against a specific antigen oreffectiveness against a spectrum of antigen sub-classes or othercriteria.

[0074] Human antibodies are excellent immunogens when injected intomice. The antigenic sites on the human antibody molecules are spreadright across the length of the molecule from the NH₂ terminii—ie the Fabend to the carboxylic acid terminus—ie the Fc end. The Fab NH₂ endcarries the paratope. Other antigenic components of the Fab are presentfor structural or “carrier” purposes. For the purposes of the vaccinethe Fc exclusively exhibits “carrier” as opposed to paratope antigens.

[0075] Immunization studies have demonstrated that not all the antigenicsites on the intact human immunoglobulin molecule are of equal value inthat a greater proportion of induced antibodies tend to be directedagainst the Fc region. This phenomena is described as antigeniccompetition or more accurately as intramolecular antigenic competition.When developing an anti-paratopic antibody, however, the part of themolecule of most interest is the Fab area that is to say the paratopebearing region. A simple way to overcome the problem of Fc dominance isto enzymatically cleave the immunoglobulin molecule and isolate the Fabfragment. When used to immunize a mouse this will cause all the inducedimmunoglobulins to be directed against the Fab fragment. A subset of theanti-Fab antibodies generated by the mouse, irrespective of whether anintact immunoglobulin molecule or a Fab/F(ab)′2 fragment has been used,will be directed against the internal idiotope i.e. paratopic image ofthe human immunogen. Thus, the member of the anti-HIV prototypic setused as an immunogen in the mouse may be either (a) the mixed intacthuman immunoglobulin specific for the AIDS virus, (b) selected classesor sub-classes of the intact immunoglobulin, (c) a Fab/F(ab)′2 fragmentof one or a combination of the AIDS specific immunoglobulins or (d) aFab/F(ab)′2 fragment of one or a combination of the AIDS specificimmunoglobulins complexed to carries eg. Keyhole Limpet Haemocyanin orhuman abumin.

[0076] The stage of preparation of immunogen may thus include enzymaticdigestion or chemical cleavage of the human anti-HIV immunoglobulin andconjugation of the Fab/F(ab)′2 to microspheres or the like.

[0077] As will be apparent from the foregoing, it is conceivable thatwhen injected into the mouse, the HIV IgG1 subgroup could provide allthe relevant paratopes on one type of carrier. This regime would favourthe generation of anti-idiotypes in the mouse (as opposed to thegeneration of anti-“carrier” molecules).

[0078] There is a possibility though of inter-molecular antigeniccompetition so that only a small variety of the human paratopes directedagainst HIV will end up being antigenic in the mouse. If this occursthen there are various ways of proceeding:

[0079] (i) after the screening step those paratopes that are dominantcould be isolated from the immunogen population and a secondimmunization carried out to develop mouse anti-paratope antibodies tothe remaining paratopes.

[0080] (ii) the anti-idiotypes/idiotypic reagents arising from the firstimmunization could be screened and tested to see if theanti-idiotypes/idiotypic reagents cover the known HIV antigens/antigenicfragments. If all the known antigens are covered by the generation ofanti-idiotypes/idiotypic reagents then a second immunization protocolmay not be necessary.

[0081] (iii) A different mouse strain could be employed. The eventualmanufacturing route thus depends on whether anti-idiotypes/idiotypicreagents to all reagents are required.

[0082] It may suffice to have, say, one or two of the haptens from eachantigen group covered. To a great extent though, this is an issue thatwill be resolved by the mouse itself in that it may only be able toraise anti-idiotypes/idiotypic reagents against a restricted idiotyperange.

[0083] How all these factors are weighted will determine the nature ofthe immunogen that is preferred for injection into the mouse.

[0084] Thus it may be preferable

[0085] (i) to choose a particular class/sub-class of HIV+ve humanimmunoglobulin which expresses several specificities and use this toimmunize the mouse.

[0086] Alternatively

[0087] (ii) given the diversity of the immunoglobulin response theantibody range may not be restricted and a more general immunizationroutine adopted. In the latter case.

[0088] (iii) sub-class purification may be called for coupled to severalprimary immunizations.

[0089] An excellent starting position though would be to opt for (i) andthen remove the Fc prior to further development of the immunogen bylinking it to adjuvants such as precipitated immunoglobulins ormicrospheres.

[0090] While stating a preference for (i) an outline of the variousalternative pathways for the purification and preparation of theimmunogen is shown if FIGS. 5 and 6.

[0091] After injection of the immunogen into mice, preferably after asecond immunization, mouse spleen cells are harvested by normal methodsand fused to NSI in accordance with conventional hybridoma technology.

[0092] Hybrids are then grown and screened and positive hybrids clonedand re-tested. The clones are then adapted and grown in serum-free mediaand specific antibodies purified and ready for testing in human.

[0093] The monoclonal antibody pool may be generated using for examplethe standard method or the “LOTTO” method as outlined in Table 3 below:TABLE 3 STANDARD METHOD “LOTTO” one-hit (multi-chance) (i) immunize (ii)4 wks later boost (ii) 2-3 days later boost (iii) 4 days later (ii) 2-3days later spleen spleen cell prepn cell prepn (iv) Hybridoma productn(iii) Hybridize (v) Preliminary screen (iv) Preliminary screen (vi)CLONE (v) Clone

[0094] The Fab pool may be screened by conventional means as shown inTable 4: TABLE 4 SCREENING FOR FAB POOL Mc Ab Configuration (+Ve/−Ve)Antigen 1 2 3 4 5 6 Bence-Jones + − + − + − Human Ig + + − + − −Immunogen − − + + − + ACTION Discard/Retain D D D D D R

[0095] The anti-idiotype pool may be screened by conventional means. Forexample, HIV on a tray is mixed with human anti-HIV antibodies beforeand after incubation with mouse HIV idiotype complexed tomicrosphere/eupergit spheres, then chased with anti-mouse Ig-PO, +Ve isdiscarded, and −Ve is retained.

[0096] Alternatively, HIV on beads is mixed with human anti-HIVPO-enzyme+mouse HIV idiotype +Ve response is discarded.

[0097] As will be appreciated by those skilled in the art the antibodiesmay be selected from a pool occurring in a different species ofvertebrate and the prototypic set may be selected from effectivenessagainst a different antigen. The antibodies may not be free in plasmabut may be bound to cells (e.g. B cells) or may exist as immune complex.The prototypic set may be divided into members using different criteriafrom that exemplified.

[0098] Other methods may be used for separation such as use of dyesbound to inert supports, or the use of monoclonal antibodies, etc. andpurification of the immunogen without departure herefrom.

[0099] The immunogens, or fragments thereof may be utilized in hostspecies other than mice.

[0100] The antibodies so obtained may be used in various ways forexample for immunization of the vertebrate from which the antibodieswere obtained, in test methods and for other purposes.

[0101] The invention will now be described more specifically by way ofthe following Examples.

[0102] Preparation of Human Immunodeficiency Virus Specific HumanAntibodies

[0103] The desired antibody is designated as mouse Ab2. However, theproduction of mouse Ab2 is dependent upon the prior availability of afirst antibody (Ab1) of interest. Presently, there are two sources ofAb1. The first source is the mouse Ab1 produced in vitro, and the secondis the human Ab1 to HIV.

[0104] There are several good reasons to choose the human Ab1 as theimmunogen. Little is known about the epitopes that are relevant in thisdisease (HIV), other than the identification of the CD4 antigen on the Tcell, and those epitopes on the envelope. It has also been postulatedthat transmembrane proteins may be of importance based on hydrophilicityindices.

[0105] The issue about epitopes is that they are primarily linearsequences. Little is currently known about conformational epitopes,neotypes or cryptotopes. A prior knowledge of epitopes of interest isunimportant when human antibodies are being used.

[0106] Secondly, the virus is known to be specific for man which is agood reason to use antibodies induced in situ as the starting materialto induce the production of the Ab2. It is possible that there arespecific epitopes which are of some importance to the human situationwhich will not be seen in other species.

[0107] A. Preparation and Purification of HIV Antigens.

[0108] Native and recombinant antigens can be purified by affinitychromatography using human antibodies or antibodies from another speciessuch as mouse monoclonal antibodies specific for the HIV antigens. Byway of illustration the procedure described will be that using humanantibodies. There is very little difference between the two approachesthough the benefit is that with the appropriate mouse monoclonalantibodies specific antigens can be purified only if the antigen sourceis the native one. However, if the antigen source is a recombinant onethen human antibodies will allow for the specific purification of therecombinant antigen. When human antibodies are used the steps involvedare

[0109] (1) the preparation of human IgG from HIV infected individuals.

[0110] (2) the preparation of the human antibody (IgG) column and

[0111] (3) the purification of the viral antigens using theaforementioned column.

[0112] 1. Preparation of Human Antibodies.

[0113] According to this procedure human antibodies were first purifiedby either hydroxyapatite chromatography, ion-exchange chromatography(DEAE-cellulose) or protein-A affinity chromatography. By way of examplethe method for the purification described is that of protein-A agarosecolumn chromatography.

[0114] Pooled human sera was obtained from patients positive for theAIDS virus as determined by both an AIDS antibody ELISA assay andsubsequently confirmed by the Western Blot assay. Prior to use the serumhad been heat treated (56° C. for 30 mins). A 2.0 ml protein-A agarosecolumn was washed with 20 ml of the Monopure binding buffer (Pierce). 4mls of the pooled serum was diluted with 8 mls binding buffer andcentrifuged (200×g:10 min:RT). The supernatant was applied to thecolumn, allowed to percolate through and exhaustively washed in thebinding buffer. The human IgG was specifically eluted using thecommercially obtained elution buffer (Pierce). Following dialysis andconcentration, the A₂₈₀ data was used to determine the concentration ofprotein which was calculated to be 30 milligrams as determined by theE^(1%)=1.43 (280 nm). Western Blot and ELISA data confirmed the presenceof HIV specific antibodies in the IgG fraction purified in this manner.

[0115] 2. The Preparation of the IgG Affinity Column.

[0116] 30 mgs of the human IgG was equilibrated in the coupling buffer(0.1M NaHCO₃ pH8.3+0.5M NaCl) and mixed with 4 gms CnBr-Sepharose 4B(Pharmacia) which had been pre-washed in 1 mM HCl, swollen andequilibrated in the coupling buffer. The mixture was mixed end-over-endin a sealed coupling vessel (2 hrs, RT). Unreactive groups on the matrixwere blocked using 0.2M glycine in the coupling buffer (16 hrs, 4° C.)and the ensuing IgG-Sepharose matrix exhaustively washed in high saltand variable pH buffers prior to the purification of the HIV antigens.

[0117] 3. The Purification of the Native/Recombinant Antigens.

[0118] By way of illustration, the method described is that for therecombinant HIV antigens in particular recombinant ‘gp120’.

[0119] Sub genomic clones of HIV cDNA encoding gp120, gp41, p24 and p18were cloned and amplified in E. coli using λ gt11. The E. Coli lysateswere screened with in-house and by commercial HIV antigen ELISA's.

[0120] Radioimmunoprecipitation studies confirmed the presence ofrecombinant HIV antigens and the molecular weights of the recombinantantigens were as predicted e.g. 60 kD for the recombinant ‘gp120’.

[0121] Following precipitation of E. Coli antigens with (NH₄)₂SO₄ thesupernate was concentrated (Amicon)dialysed against distilled water andthen against 0.05M Phosphate buffer pH7.2(16 hrs, 4° C.). 40 mls of thedialysed concentrate was combined with approximately 2 ml of theIgG-Sepharose and the mixture incubated end-over-end for 2 hrs(RT). Thematrix was exhaustively washed and the recombinant protein eluted using4M MgCl₂, pH 8.3. The presence of recombinant antigen was confirmed asoutlined above.

[0122] B. The Purification of the Human HIV Specific Antibodies.

[0123] The purification of HIV specific human antibodies involved twosteps. These are outlined below.

[0124] i The preparation of the HIV antigen column.

[0125] ii The preparation of the HIV specific human antibodies.

[0126] 1. Preparation of the HIV antigen-Sepharose column.

[0127] 7.5 mls of the eluted protein was mixed with 2 gms swollen,pre-washed and appropriately equilibrated CnBr-Sepharose (pH8.3). Themixture was mixed end-over-end (2 hrs, RT). Unreactive sites wereblocked using 0.2M glycine (16 hrs, 4° C.) and the matrix exhaustivelywashed as outlined for the IgG-Sepharose column.

[0128] 2. Purification of HIV Specific Human Antibody.

[0129] 4 mls of pooled human HIV serum heat treated as outlined abovewas passed through a PD-10 column equilibrated with freshly prepared0.05M Phosphate buffer pH7.2+0.5M NaCl. The first 3 mls fraction (voidvolume) was discarded and the next 7.5 mls was collected. 10 mls of thegp120-Sepharose matrix and 7.5 mls of the equilibrated serum were mixedend-over-end for 2 hrs at RT. Following extensive washing HIV specificIg's were desorbed using buffer containing 4M MgCl₂ pH8.3.Approximately, 2 mg of HIV specific Ig was obtained using this method.

[0130] C. The Production of Mouse Monoclonal Antibody to the Human AB1.

[0131] The production of Mouse monoclonal antibodies firstly involvesthe induction of the antibodies either by in vivo methods or by in vitromethods.

[0132] By way of illustration the in vitro method is described.

[0133] Two groups of Balb/c mice were used in this experiment. The firstgroup consisted of mice which had been tolerized to human IgG1. This hadbeen achieved by injecting mice intraperitoneally, 7 days previously,with 10 milligrams of human IgG1. The second group consisted ofuntolerized mice.

[0134] Mouse Ab2 antibodies were induced in the following way. 1.3×10⁸mouse spleen cells were recovered and washed in the incubation medium(Iscoves DMEM medium containing 20% foetal calf serum (FCS), 40% thymusconditioned medium (TCM), 5×10⁻⁴ 2-mercaptoethanol, 4 mML-glutamine 50IU Penicillin and 50 IU streptomycin). HIV specific humanimmunoglobulins at a concentration of 10 micrograms/ml incubation mediumwas added to the mouse spleen cells. The total volume used in theincubation of the spleen cells with human antibody varied between 10 and15 mls. In this example, the incubation was allowed to proceed for 7days in a heated (37° C.) CO₂ incubator.

[0135] Following incubation the cells were recovered for fusion to etherSP2, NS1 or X63-Ag*.653 mouse myeloma cells. The viability of the spleencells was found to vary between 70 and 99% and the viability of themyeloma was generally 99%. For the sake of illustration SP 2 mousespleen cells were used though other cells such as rat or human myelomacells could be used in this procedure. Spleen cells were fused to themyeloma cells using polyethylene glycol 1500/4000 (Boehringer/Mannheim)using standard procedures and following 24 hrs incubation in a CO₂incubator at 37° C. the hybrids were plated out in the incubation mediumnow containing HAT.

[0136] Screening of Hybrid Supernates for Ab2 Specificity

[0137] Screening for Ab2 was carried out by ELISA. Normal human IgG wascoated onto the ELISA trays (1 μg/ml in carbonate-bicarbonate buffer pH9.6, 4° C., 16 hrs). HIV specific immunogen was coated (0.6 μg/ml) on aseparate tray, and mouse supernatants were added in the usual way. Trayswere blocked with 2% BSA in PBS-Tween 20 and the color was allowed todevelop using ABTS substrate. HIV IgG was also fragmented using Pepsinbeads and the trays coated with the F(ab)′2 to further determine thespecificity of the mouse Ab2.

[0138] In the screening protocol, antibodies obtained from uninfectedindividuals and HIV-infected individuals were coated onto separate ELISA(screening) plates. If mouse Ab2 bound to both plates it was discarded.If it bound only to the plate containing normal human antibody, it wasalso discarded. If the Ab2 bound the HIV plate exclusively it was usedas a vaccine candidate. Several such vaccine candidates (Ab2) werefound.

[0139] D. Recovery of Anti-HIV Antibody Clones Produced in situ as aResult of Natural Infection.

[0140] In addition to the serum HIV antibodies purified by theabovementioned methods it is possible to obtain the human Ab1 byEpstein-Barr virus (EBV) transformation of human B cells obtained fromindividuals exposed to the AIDS virus.

[0141] By way of illustration the following method was used.

[0142] Human peripheral blood lymphocytes (PBL's) were diluted 1:1 inphosphate buffered saline and the red cells removed by centrifugationthrough a Ficoll-hypaque cushion (pharmacia).

[0143] The PBL's either depleted or not depleted of monocytes andlymphocytes using methods familiar to those skilled in the art, werethen transformed using for example the EBV isolate B95-8 in steriletissue culture media (RPMI-1640)+5% FCS). In a simple example the B95-8isolate is made available as a supernate which is mixed with themonocyte/T cell depleted fraction enriched for the B lymphocytes. Thecells are grown in this mixture, fed as required, and expanded in96-well flat bottomed plates prior to fusion with the mouse myeloma cellline such as X63-Ag*.653. Screening is by a commercially available HIVantibody ELISA. Cloning and feeding (Medium containing HAT/HT) is by theusual method except that non transformed will be selected out by feedingwith 1 micromolar Oubain.

[0144] All these methods must be carried out in hybridoma facilitiessuitable for work involving HIV as virus may be shed under theseconditions.

[0145] E. Production of Human AB1 Using in Vitro Immunization of HumanPeripheral Blood Lymphocytes and/or Splenic Lymphocytes.

[0146] HIV specific human Ab1 may also be obtained by in vitroimmunization using whole virus or native, recombinant HIV antigens andantigens bound to nitrocellulose. According to one method 3-4×10⁴ humanPBL's or human splenic lymphocytes depleted of monocytes/T lymphocytesusing L-Leucine methyl ester can be immunized with small amounts (1nanogram-10 micrograms) of HIV antigen. The human Ab1 are monoclonalwhen the techniques of hybridoma technology as outlined in D. are used.Human. Ab1 obtained in this way may be used as the immunogen to producethe Ab2 by either in vivo or in vitro culture techniques using humancells or cells of other species as the human Ab1 would house theprototypic paratopes as defined by the foregoing.

[0147] Such variations as will be apparent to those skilled in the artfrom the teaching hereof are deemed to be within the scope of theinvention herein disclosed.

1-16. Cancelled.
 17. A method of manufacture of an anti-paratopicantibody comprising the steps of: (i) selecting from a pool ofantibodies occurring in a first species of vertebrate a prototypic set,wherein members of the prototypic set are antibodies which bind to anaetiological agent of interest, wherein said aetiological agent ofinterest is capable of causing a mammalian infection; (ii) subdividingthe prototypic set selected in step (i) into antibody classes IgG, IgA,IgM, IgD and IgE; (iii) screening the antibody classes from step (ii) toselect one or more classes which bind a specific antigen or group ofantigens expressed by the aetiological agent of interest in the firstspecies of vertebrate; (iv) utilizing one or more of the antibodyclasses selected in step (iii) as an immunogen in a host of a differentspecies from the first species or in an in vitro incubation systemcomprising spleen cells derived from the same or a different speciesfrom the first species, to produce antibodies having a characteristicwhich are anti-paratopic with respect to said immunogen and which are asynthetic replicate of the specific antigen or group of antigens used instep (iii); and (v) selecting, isolating and purifying theanti-paratopic antibodies produced in step (iv).
 18. The methodaccording to claim 17 wherein the antibody classes prepared in step (ii)are further subdivided into their immunoglobulin subclasses.
 19. Themethod according to claim 17 wherein antibodies from the antibodyclasses selected in step (iii) are subjected to enzymatic cleavage toseparate F(c) and F(ab) antibody fragments and the F(ab) antibodyfragments are used as the immunogen in step (iv) of the method.
 20. Themethod according to claim 17, wherein the aetiological agent is HIV. 21.The method according to claim 20, wherein the specific antigen or groupof antigens employed in step (iii) is selected from the group consistingof p18, p24, gp41, p55, gp120 and gp160.
 22. The method according toclaim 17 wherein spleen cells selected in step (iv) are harvested andfused to myeloma cells to produce a hybridoma cell line which is capableof producing an anti-paratopic antibody.
 23. The method according toclaim 22 wherein the antibodies produced are monoclonal antibodies. 24.The method according to claim 22 wherein the antibodies produced arepolyclonal antibodies.
 25. Purified non-human anti-paratopic antibodiesproduced according to the method of claim
 17. 26. Purified non-humananti-paratopic antibodies produced according to the method of claim 20.27. Purified non-human anti-paratopic antibodies produced according tothe method of claim
 21. 28. The method according to claim 17, whereinthe antibody classes of step (ii) are further subdivided into subclassesprior to performing step (iii) and wherein in step (iii) one or moresubclasses are selected.
 29. The method according to claim 28 whereineach subclass of antibody exhibits a level of immunological activity andwherein in step (iii) the selected subclass is that one with the highestlevel of immunological activity of the subclasses present.
 30. Themethod according to claim 17 wherein each class of antibody exhibits alevel of immunological activity and in step (iii) the selected class isthat one with the highest level of immunological activity of the classespresent.